Method and Materials for Quaternary Amine Catalyzed Bisulfite Conversion of Cytosine to Uracil

ABSTRACT

The invention provides methods and materials for the conversion of cytosine to uracil. A nucleic acid, such a gDNA, is reacted with bisulfate, such as magnesium bisulfite, in the presence of a quaternary amine catalyst. Examples of suitable quaternary amine catalysts include but are not limited to quaternary ammonium compounds, quaternary alkyl ammonium salts, quaternary alkyl ammonium halides, quaternary methyl ammonium bromide, quaternary ammonium chloride, tetraethyl ammonium hydroxide, tetraethylammonium chloride, tetrabutyl ammonium chloride, tetrabutyl ammonium bromide. The invention also contemplates kits of premeasured ingredients for carrying out the methods of the invention either on an individual sample or on a plurality of samples.

This application is a continuation of application Ser. No. 10/926,528,filed Aug. 26, 2004 and claims benefit of priority to U.S. ProvisionalApplication Ser. Nos. 60/499,106 filed Aug. 29, 2003 and 60/523,054filed Nov. 17, 2003, each of which is hereby incorporated by reference.

FIELD

The invention relates generally to methods and materials for theconversion of cytosine to uracil.

BACKGROUND

Assessment of methylation of DNA is useful in many research, diagnostic,medical, forensic, and industrial fields. Key to this assessment isconverting cytosine, but not methylcytosine, to uracil, but not thymine.One basic method for such conversion, employing sodium bisulfite, iswell known. Over the years, the method has been improved in attempts toovercome disadvantages that include tedious procedures, lengthy reactiontimes, and DNA degradation. The most commonly used protocol is taught byJ. Herman, Proc. Natl. Acad. Sci. 93, 9821-26 (1996), incorporatedherein by reference in its entirety. This method involves denaturation,reaction with sodium bisulfite in the presence of hydroquinone, andsubsequent completion of the modification by treatment with NaOH.Despite the attempts to improve the protocol, current procedures requirepre-denaturation of the genomic DNA (gDNA) to single stranded DNA(ssDNA), preparation of fresh solutions of sodium bisulfite (NaHSO₃),typically about 3M, and inclusion of an anti-oxidant (e.g.,hydroquinone). The protocol also requires long reaction times andtedious clean-up procedures.

In addition, the currently employed sodium bisulfite protocols areplagued by reports of incomplete conversion, irreproducible results, andother problems. In some cases, the reaction can result in significantDNA degradation (reportedly as high as 96%), making it difficult toobtain enough sample for further analysis. See. S. J. Clark et al.Nucleic Acid Research 2001, 29 no. 13, e65. Given the importance ofassessment of DNA methylation, it can be seen that there is a need forimproved methodologies for conversion of cytosine to uracil.

It has been discovered that bisulfite methods that employ magnesiumbisulfite, polyamine compounds, and/or quaternary amine compoundsprovide useful alternatives to sodium bisulfite conversion reactions.These discoveries are the subjects of co-owned applications entitled“Method And Materials For Polyamine Catalyzed Bisulfite Conversion OfCytosine To Uracil” (U.S. application Ser. No. 60/499,113 filed Aug. 29,2003, and also application Ser. No. 60/499,113 (docket no. 5065P2)having the same title and filed Nov. 17, 2003), “Method And MaterialsFor Quaternary Amine Catalyzed Bisulfite Conversion Of Cytosine ToUracil” (U.S. application Ser. No. 60/499,106 filed Aug. 29, 2003, and“Method and Materials for Bisulfite Conversion of Cytosine to Uracil”(U.S. application Ser. No. 60/499,082 filed Aug. 29, 2003, and alsoapplication Ser. No. 60/523,056 having the same title and filed Nov. 17,2003), all of which are hereby incorporated by reference in theirentirety Improvements in clean-up procedures associated with conversionof cytosine to uracil are also the subject of co-owned applicationsentitled “Improved Bisulfite Method” (U.S. application Ser. No.60/498,996 filed Aug. 29, 2003, and also application Ser. No. 60/520,941(5109P2) having the same title and filed Nov. 17, 2003) all of which arehereby incorporated by reference in their entirety.

SUMMARY

In certain embodiments of the invention, the invention comprises methodsof specifically converting cytosine to uracil by using a catalyzedbisulfite reaction.

In some embodiments, the present invention provides methods for theconversion of cytosine to uracil in a nucleic acid comprising the stepsof:

reacting a nucleic acid comprising at least one cytosine nucleobase withbisulfite ion in the presence of a quaternary amine catalyst.

In some embodiments, the quaternary amine comprises a compound havingFormula I:

or a derivative thereof, wherein:

R₁, R₂, R₃ and R₄ are each independently alkyl, preferably C₁-C₄ alkyl;and

Z^(θ) is selected from the halides and OFF.

In some embodiments, R₁, R₂, R₃ and R₄ are identical.

In some embodiments of the invention, the quaternary amine catalystcomprises a quaternary ammonium compound, or a derivative thereof. Infurther embodiments, the quaternary amine catalyst comprises aquaternary alkyl ammonium salt. In yet further embodiments, thequaternary amine catalyst comprises a quaternary alkyl ammonium halide,for example a quaternary ammonium chloride or a quaternary ammoniumbromide. In some embodiments, the quaternary amine catalyst comprises atleast one of quaternary methyl ammonium bromide, tetraethyl ammoniumhydroxide, tetraethylammonium chloride, tetrabutyl ammonium chloride andtetrabutyl ammonium bromide.

In some embodiments, the reaction of the nucleic acid and bisulfite ionis performed in a solution containing bisulfite ion, such as magnesiumbisulfite, in a concentration of from about 0.5M to about 2.5M. Infurther embodiments, the solution contains bisulfite ion, such asmagnesium bisulfite, in a concentration of from about 1M to about 2M.

In some embodiments, the magnesium bisulfite is present at aconcentration of at least about 1M.

Also provided are methods for the conversion of cytosine to uracilcomprising the steps of reacting a DNA sample in solution with abisulfite salt and quaternary amine catalyst as described above, whereinthe concentration of the bisulfite salt is from about 0.5M to about 2M.In further embodiments, the concentration of the bisulfite salt is about1.3M.

In some embodiments of the methods of the invention, the reaction isperformed at a temperature from about 40 to about 60 degrees, such asabout 50 degrees, for about 4 to about 15 hours. In further embodimentsof the methods of the invention, the nucleic acid is gDNA.

Also provided in accordance with the present invention are kits for usein conversion of cytosine to uracil comprising magnesium bisulfite; anda quaternary amine catalyst as described above. In some embodiments ofsuch kits, the magnesium bisulfite is provided as an approximately 2Mmagnesium bisulfite solution. In further embodiments of the kits of theinvention, the quaternary amine catalyst comprises tetraethyl ammoniumhydroxide. In some embodiments, the kits further comprise reagents forsequencing and/or amplification (e.g., by PCR), for example a polymeraseand one or more primers. In some embodiments, the kits containpremeasured materials useful in various embodiments of the methods ofthe invention.

In some embodiments, the methylation status of one or more cytosines inthe target nucleic acid(s) can be determined by any suitable method.Typically, methylation status can be determined by measuring thepresence or relative amount of uracil at a nucleotide position that waspreviously non-methylated cytosine, and was converted to uracil by thebisulfite treatment. If desired, the presence or relative amount ofresidual cytosine at the same nucleotide position (indicating thepresence of methylcytosine) can be measured for comparison with theamount of uracil, to determine the degree of methylation at theparticular nucleotide position. Appropriate control experiments can alsobe performed to correct for incomplete transformation of cytosine touracil, if desired.

The presence or amount of uracil and/or methylcytosine at a particularnucleotide position can be measured by any suitable method, such as DNAsequencing (e.g., by the Sanger method or Maxam-Gilbert method orsubsequent embodiments thereof (e.g., using dye-labeled terminators ordye-labeled primers, such as discussed in WO 02/30944 and by Ansorge etal. DNA Sequencing Strategies—Automated and Advanced Approaches, JohnWiley & Sons, New York, 1997)), PCR (e.g., primer-specific PCR),oligonucleotide ligation assay (OLA) or other ligation-dependenttechniques (e.g., see U.S. Pat. No. 6,511,810 and references citedtherein), single base extension (over the potential methylation site),mass spectrometry, real time PCR (e.g., using labeled probes that arecomplementary to C and or U), microarrays comprising sequence specificprobes, etc. Various exemplary techniques are also described by Kirk etal., Nucl. Acids Res., 30:3295-3311 (2002).

DETAILED DESCRIPTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed. In thisapplication, the use of the singular includes the plural unlessspecifically stated otherwise. In this application, the use of “or”means “and/or” unless stated otherwise. Furthermore, the use of the term“including”, as well as other forms, such as “includes” and “included”,is not limiting. Also, terms such as “element” or “component” encompassboth elements and components comprising one unit and elements andcomponents that comprise more than one subunit unless specificallystated otherwise.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including but not limited to patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference intheir entirety for any purpose.

DEFINITIONS

As used herein, the term “alkyl” is intended to mean saturatedhydrocarbon species, including without limitation straight, branchedchain and cyclic hydrocarbons, for example, methyl, ethyl, n-propyl,isopropyl, n-butyl, sec-butyl, t-butyl, n-pentyl, sec-pentyl, t-pentyl,neopentyl, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl groups.

As used herein, the term “EXO/SAP” denotes a mixture of exonuclease I(EXO) and shrimp alkaline phosphatase (SAP).

As used herein, the term “gDNA” refers to genomic DNA.

Bisulfite ion has its accustomed meaning of HSO₃ ⁻. Typically, bisulfiteis used as an aqueous solution of a bisulfite salt, for examplemagnesium bisulfite, which has the formula Mg(HSO₃)₂, and sodiumbisulfite, which has the formula NaHSO₃.

The term “PCR” is intended to denote polymerase chain reaction, as iswell known in the art. The term “MSP” denotes methylation specific PCR,such as described by J. Herman, Proc. Natl. Acad. Sci. 93, 9821-26(1996), and modified as discussed herein.

As used herein, the term “nucleic acid” includes, for example,nucleobase-containing polymeric compounds, including naturally occurringand non-naturally occurring forms thereof, for example and withoutlimitation, genomic DNA, cDNA, hnRNA, mRNA, rRNA, tRNA, fragmentednucleic acids, nucleic acids obtained from subcellular organelles suchas mitochondria or chloroplasts, and nucleic acids obtained frommicroorganisms, or DNA or RNA viruses that may be present on or in abiological sample.

The term “quaternary amine compound” or “quaternary amine” is intendedto include, without limitation, compounds containing a tetra-substitutednitrogen atom, and the salts and hydroxides of such compounds. Examplesof quaternary amine compounds include, without limitation, quaternaryalkyl ammonium compounds, for example quaternary alkyl ammonium halides.Thus, quaternary amine compounds include quaternary alkyl ammoniumchlorides such as quaternary methyl ammonium bromide, quaternary alkylammonium bromides, quaternary ammonium chlorides, tetraethyl ammoniumhydroxide, tetraethyl ammonium chloride, tetrabutyl ammonium chloride,tetrabutyl ammonium bromide, and the like.

The term “ssDNA” refers to single stranded DNA, resulting typically, butnot exclusively, from denaturing double stranded DNA (“dsDNA”).

The term “TE buffer” refers to the well-known buffering solution of 10mM TRIS-HCl and 1 mM EDTA that is typically used in analysis of nucleicacids.

The term “triamine” refers to compounds having three amino groups,including but not limited to diethylene triamine (DETA), guanidine HCl,tetramethyl guanidine, and the like.

The term “nucleic acid sample” is intended to denote a sample (e.g., acomposition, mixture, suspension or solution) that contains at least onenucleic acid.

Unless otherwise specified, reference herein to cytosine refers tounmethylated cytosine and conversion refers to specific conversion ofunmethylated cytosine to uracil.

All reported temperatures are in degrees Celsius unless statedotherwise.

In some embodiments, the present invention provides methods ofconverting cytosine to uracil in a nucleic acid sample by using acatalyzed bisulfite reaction. The methods of the present invention canprovide significant benefits.

The nucleic acid samples may be obtained by any conventional collectionand purification process prior to use in the methods of the invention.The examples discussed below used commercially available sample lines(e.g. from Coriell or Intergen) of known methylation status, to assessthe viability of the methods.

Typically, the product of the reaction between the nucleic acid andbisulfite is reacted with a base to complete the conversion of cytosineto uracil. One typical base is NaOH. In some embodiments the methodsherein further comprise the step of purifying the bisulfite-reactednucleic acid prior to treatment with base. In some further embodiments,the methods further comprise the step of analyzing the product of thebisulfite conversion reaction, for example by mass spectrometry, toconfirm completion of the bisulfite conversion reaction.

Typical protocols in the art require the use of 3M sodium bisulfite,long reaction times of up to 16 hours, and the presence of anantioxidant. Because of the relatively high salt concentration, the lowpH of the reaction and the long reaction times, the DNA can be degraded.Additionally, the ss DNA resulting from the gDNA is difficult to purifyaway from the high salt concentration used in the reaction. In addition,it is typically necessary to remove most of the bisulfite, whichinterferes with subsequent enzymatic reactions, for example those of PCRprotocols. Prior procedures also require freshly prepared solutions ofbisulfite and antioxidant (typically hydroquinone).

Embodiments of the methods of the present invention may overcome one ormore disadvantages of prior methods. For example, it has been discoveredin accordance with the some embodiments of the present invention thatthe reaction of a nucleic acid of interest with bisulfite ion, such asmagnesium bisulfite, in the presence of a quaternary amine in accordancewith the methods disclosed herein afford faster reaction times. Inaddition, because the reaction time is faster, less oxidation may occur.Thus, the presently disclosed methods do not require addition of anantioxidant such as hydroquinone. Additionally, magnesium bisulfitesolution at 1M concentration may remain acidic in the presence ofeffective concentrations of polyamine catalyst (for example 0.1M DETA),whereas the corresponding solution of sodium bisulfite salt does not.Thus, methods of the invention can employ bisulfite concentrations thatare significantly less than the methods known in the art, therebyaffording facilitated sample preparation for PCR. Moreover, it has beendiscovered herein that stock magnesium bisulfite solutions can beemployed, thus eliminating the need to freshly prepare those solutions.Finally, methods of the invention reduce or eliminate the need for aseparate predenaturation step, and can be performed in a greatly reducedreaction volume. Thus, methods of the present invention can afford PCRyields similar to those of protocols previously known in the art, butwith reduced preparation times, reaction times, and clean-up efforts.

Suitable counter-ions for the bisulfite compound may be monovalent ordivalent. Examples of monovalent cations include, without limitation,sodium, lithium, potassium, ammonium, and tetraalkylammonium. Suitabledivalent cations include, without limitation, magnesium, manganese, andcalcium.

In certain embodiments, the invention comprises kits for carrying outthe methods of the invention. In one embodiment, a kit of the inventionincludes pre-measured ingredients required for carrying out thebisulfite reaction, such as magnesium bisulfite and catalyst. In certainembodiments, the catalyst comprises tetraethyl ammonium hydroxide. Incertain embodiments, the invention includes a kit containingpre-packaged materials sufficient to prepare multiple samples. In yetanother embodiment, the materials will be pre-packaged with appropriateEppendorf tubes or other reaction vessels, as appropriate.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including but not limited to patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference intheir entirety for any purpose.

The examples described herein are certain embodiments chosen toillustrate the invention. Applicant does not limit the invention tothese embodiments. Rather, Applicant acknowledges that those reasonablyskilled in the art will readily recognize additional variants that donot differ from the scope and spirit of the inventions disclosed herein.

EXAMPLES

In accordance with the present invention, it has been found thatquaternary amines, such as tetraethylammonium hydroxide (ET₄NOH), areuseful catalysts in the bisulfite conversion of cytosine to uracil innucleic acid samples.

Methyl-Specific PCR Analysis

Each sample discussed herein was analyzed by methyl-specific PCR (MSP).MSP provides a relatively fast analysis method for methylation status ofbisulfite-treated DNA samples, providing a yes/no answer. The method isbased on using primer pair sets. One primer pair is designed toanneal/PCR amplify only if all cytosines were successfully converted touracil, and the other primer pair in the set annealed/PCR amplified ifthe methylated cytosine (CpG cytosines only) were methylated, andtherefore not converted to uracil.

The MSP pairs that amplify specific gene fragments, and the expectedsize of the amplicon, are the following:

for the p16 gene, unmethylated reaction (size 151):

5′-TTATTAGAGGGTGGGGTGGATTGT-3′ (sense), 5′-CAACCCCAAACCACAACCATAA-3′(antisense);

methylated reaction (size 150):

5′-TTATTAGAGGGTGGGGCGGATCGC-3′ (sense), 5′-GACCCCGAACCG-CGACCGTAA-3′(antisense);

for the MGMT gene, unmethylated reaction (93):

5′-TTTGTGTTTTGATGTTTGTAGGTTTTTGT-3′ (sense),5′-AACTCCACACTCTTCCAAAAACAAAACA-3′ (antisense);

methylated reaction (81):

5′-TTTCGACGTTCGTAGGTTTTCGC-3′ (sense), 5′-GCACTCTTCCGAAA-ACGAAACG-3′(antisense);

for the DAP-kinase gene, unmethylated reaction:

5′-GGAGGATAGTTGGATTGAGTTAATGTT-3′ (sense), 5′-CAATCCCT-CCCAAACACCAA-3′(antisense);

methylated reaction:

5′-GGATAGTCGGATCGAGTTAACGTC-3′ (sense), 5′-CCCTCCCAAACGCCG-3′(antisense);

for the MLH1 gene, unmethylated reaction (124):

5′-TTTTGATGTAGATGTTTTATTAGGGTTGT (sense) 5′-ACCACCTCATCATAACTACCCACA(antisense)

methylated reaction (115)

5′-ACGTAGACGTTTTATTAGGGTCGC (sense) 5′-CCTCATCGTAACTACCCGCG (antisense)

for the p15 gene, unmethylated reaction (154):

5′-TGTGATGTGTTTGTATTTTGTGGTT (sense) 5′-CCATACAATAACCAAACAACCAA(antisense)

methylated reaction (148)

5′-GCGTTCGTATTTTGCGGTT (sense) 5′-CGTACAATAACCGAACGACCGA (antisense)

The PCR recipe used to evaluate the samples was:

2X Taq Gold PCR Master Mix  10 μL Fwd primer (5 μM)   1 μL Rev primer (5μM)   1 μL Bisulfite treated DNA 0.5 μL H2O 7.5 μL  20 μL

2×TaqGold PCR master mix is commercially available from AppliedBiosystems. The forward and reverse primer sequences are those listedabove.

The following thermal cycling schedule was used:

40 cycles 95 deg 5 min 95 deg 30 sec 60 deg 45 sec 72 deg 1:00 min  4deg forever

One of the primers in each set was synthesized with a 5′FAM label. A 1uL aliquot of the above PCR reaction was added to HiDi formamide withROX 500 size standard added, and denatured by heating at 95° C. for 5min By using a FAM-labeled primer, the PCR amplicon was directlyanalyzed on an ABI PRISM® 310 Genetic Analyzer, with POP-4™ polymer,using run module “GS POP4 (1 μL) A” (reagents and instrument all fromApplied Biosystems).

The presence of a PCR amplicon (i.e. a “peak”) having the correct sizeas observed using the 310 Genetic Analyzer indicated a successfulreaction. Additionally, the height or area of the peak could be usedempirically to determine how much template (i.e. bisulfite-treated gDNA)was initially present. The bigger the peak, the more DNA was initiallypresent.

The MSP-PCR product was then sometimes sequenced for further“resolution”. DNA sequencing was by standard protocol and reagents fromApplied Biosystems.

Prior to sequencing of the PCR amplicon, the primers and excess dNTPsused during the MSP-PCR were removed by treatment of a 4 μL aliquot ofthe PCR reaction with an equal volume mixture containing 2 Units each ofShrimp Alkaline Phosphatase (SAP) and exonuclease 1 (exo) (USBCorporation, Cleveland, Ohio). The reaction was incubated at 37° C. for1 hr, and then heat-denatured at 75° C. for 15 min. A 4 μL aliquot ofthe exo/SAP reaction was added to a solution containing 1-4 μL ofBigDye® Terminator v1.1 cycle sequencing reaction mix (AppliedBiosystems), 2 μL of BigDye® Terminator v1.1 5× sequencing buffer, 2 μLof the reverse PCR primer (5 μM) (which did not have a FAM-label), andenough water for a final volume of 20 μL. Thermal cycling: 95° C./1 min,50 cycles of 96° C./10 sec, 52° C./10 sec, 60° C./4 min, and stored at4° C. The cycle-sequencing reaction products were purified by an EdgeBiosystems Performa® 96-well plate, dried under vacuum, dissolved in 20μL of HiDi Formamide and analyzed on an ABI Prism 3730 DNA Analyzer withKB basecaller or a 3700 DNA Analyzer.

General Protocol for Quaternary Amine Conversion

A basic protocol for the quaternary amine conversion reaction is setforth below for purposes of illustration. In an Eppendorf tube, or othersuitable vessel, about 300 ng DNA, 10 μL water, 10 μL 20% Et₄NOH (toabout 0.1 mM final concentration), and 85 μL magnesium bisulfite (toabout 1.3M final concentration) are combined. The resulting mixture isincubated at about 50° for 4 to 15 hours, prior to purification andsubsequent PCR of the treated product. Purification can be conducted byexisting means, such as in accordance with the protocol of J. Herman,DNAs 93, 9821-26 (1996), incorporated herein by reference in itsentirety, or with commercially available kits such as the Wizard DNAclean-up kit (available from Promega) or the EZ DNA Methylation Kit™(available from Zymo Research).

A new purification method, which recovers the bisulfite-treated DNA veryeffectively, is the subject of the application entitled “ImprovedBisulfite Method” (U.S. application Ser. No. 60/498,996 filed Aug. 29,2003, and also application Ser. No. 60/520,941 (5109P2) having the sametitle and filed concurrently herewith) each of which is herebyincorporated by reference in its entirety.

In contrast with known sodium bisulfite conversion reaction protocols,all bisulfite conversion reactions disclosed herein were carried outwithout predenaturation, and without the inclusion of an antioxidant(e.g., hydroquinone), unless otherwise indicated.

The magnesium bisulfite used in the Examples described herein waspurchased as a 2M Mg (HSO₃)₂ solution from Aldrich Chemical Co.,Milwaukee, Wis. The pH of the solution was 2.6. The solution was usedoff-the-shelf, as purchased, and was not freshly prepared prior to eachuse.

Determination of Catalytic Effect of Quaternary Amines

Examples 1-3, shown in Table 1 below, show the catalytic effect ofquaternary amine compounds.

TABLE 1 Ex. 1 Ex. 2 Ex. 3 3 μL Coriell 3 μL Coriell 3 μL Coriell#NA09024C #NA09024C #NA09024C — 45 μL water 35 μL water 85 μL 2 M 85 μL2 M Mg(HSO₃)₂ 85 μL 2 M Mg(HSO₃)₂ Mg(HSO₃)₂ 45 μL TBAC (2M) 5.5 μL 2MDETA 10 μL 20% Et₄NOH

The resultant products were purified by standard methods (Wizard kit)and analyzed by MSP using the following MSP primer set: p15M, Dapk M,MgMt M, and p16M and four corresponding unmethylated templates: p15 U,Dapk U, MgMt U, and p16 U. The methylated primer pairs, p15M, Dupk M,Mgmt M, and p16M were all negative, as expected, since the gDNA samplewas not expected to be methylated. No product peak was expected or seenas with the p15M template. However, for the unmethylated gDNA samples,well-defined product peaks were seen in Et₄NOH and TBAC catalyzedreactions when the unmethylated primer pairs were used in MSP analysis.

Effect of Antioxidant and Concentration of Quaternary Amine

Examples 5-8, shown below in Table 2, vary with respect to the presenceor absence of antioxidant, and the identity and amount of quaternaryamine.

TABLE 2 Ex. 5 Ex. 6 Ex. 7 Ex. 8 3 μL NA 13705 3 μL NA 13705 3 μL NA13705 3 μL NA 13705 5.5 μL 2M NaOH 5.5 μL 2M NaOH 35 μL water 35 μLwater 45 μL 2M TBAC 45 μL 2M TBAC 10 μL 20% Et₄NOH 20 μL 20% Et₄NOHincubate at 37° for 10-12 incubate at 37° for 10-12 minutes minutes 55μL 2M Mg(HSO₃)₂ 30 μL Hydroquinone 85 μL 2M Mg(HSO₃)₂ 60 μL 2M Mg(HSO₃)₂— 85 μL 2M Mg(HSO₃)₂ — —

Each of Examples 5-8 were incubated at 50° for four hours. The reactionmixture in example 8 immediately formed a large amount of precipitate,which remained even after incubation, and was excluded from furtherstudy. Example 7 had a non-interfering amount of precipitate and wasretained in the study. After the four hour incubation, the remainingthree samples were purified according to the new purification methodreferred to above, which employed a size-exclusion purification process.The process uses a Microcon 100 (Millipore) size-exclusion device. Thesample and 200 μL of water were added to the Microcon 100 device, andthe sample was then spun in the device at approximately 2800 RPM forabout 8 minutes (as per manufacturers recommendation). The resultantfiltrate was removed. Two subsequent washes with about 300 μL water,each spun at about 2800 RPM for 8 minutes followed. After each, thefiltrate was again removed. About 300 μL 0.1N NaOH was added and spun atapproximately 2800 RPM for about 8 minutes. Again, the filtrate wasremoved. After addition of about 300 μL of water, the sample was spun inthe device at 2800 RPM for about 6-8 minutes. The filtrate was removedand about 50 μL TE buffer (approximately pH 8) was added. After about 5minutes before it was inverted to collect the purified DNA sample in acentrifuge. Approximately 60 μL were collected.

The bisulfite-treated DNA was analyzed by MSP using the following MSPprimer sets: p15 M, p15 U, Dapk M, Dapk U, Mgmt M, Mgmt U, p16 M, andp16 U. Hydroquinone did not appear to greatly enhance PCR yields. TheEt₄NOH sample displayed a far greater product peak than TBAC with orwithout hydroquinone. Only about 6 ng of bisulfite-treated gDNA was usedper PCR. Prior experiments using the published purification protocol(Wizard resin) provided much less isolated DNA based on the amountrequired for successful PCR. These data support the use of quaternaryamine, and specifically Et₄NOH, as a catalyst.

Reduced gDNA Concentration in Et₄NOH Catalyzed Magnesium BisulfiteReaction

The samples in Examples 9-12, shown in Table 3, demonstrate thebisulfite conversion of reduced amounts of DNA. These samples vary withrespect to either the amount of DNA used, or reduced concentration ofmagnesium bisulfite/Et₄NOH.

TABLE 3 Ex. 9 Ex. 10 Ex. 11 Ex. 12 3 μL Coriell 13705 1 μL Coriell 137051 μL Coriell 13705 1 μL Coriell 13705 35 μL water 35 μL water 35 μLwater 35 μL water 10 μL 20% Et₄NOH 10 μL 20% Et₄NOH 5 μL 20% Et₄NOH 2.5μL 20% Et₄NOH 85 μL 2M Mg(HSO₃)₂ 85 μL 2M Mg(HSO₃)₂ 40 μL 2M Mg(HSO₃)₂85 μL 2M Mg(HSO₃)₂ ~1.3M final ~1.3M final ~1.0M final ~0.8M final

Example 9 (34, of Coriell) contained about 1 μg DNA, and Examples 10-12(1 μL of Coriell) contains about 300 ng DNA. Each of these samples wasallowed to react as previously discussed, at 50° C. for four hours.Subsequent to this incubation period, each was subject to thesize-exclusion purification process discussed above, using the Microcon100 device. The process differed from that previously discussed only inthat slightly more water was used, and about 350 μL of 0.1M NaOH wasused. Each was collected in about 504, TE buffer, and 14, was used insubsequent PCR. Surprisingly, the 300 ng sample at 1.3M magnesiumbisulfite (Example 10) was observed to provide more PCR product than the1 μg sample (Example 9).

Effect of Enzyme Concentration and Template (gDNA) Concentration in MSP

The same bisulfite-treated samples above, specifically, Ex. 9 and Ex.10, were further analyzed by MSP under alternative conditions: (a)additional polymerase (TaqGold) and (b) less bisulfite-treated gDNAtemplate. The MSP conditions are shown in Table 4, below.

TABLE 4 Ex. 13 Ex. 14 Ex. 15 Ex. 16 Ex. 17 Ex. 18 Ex. 9 Ex. 10 NTC 1/10of 1/20 of Ex. 9 1/30 of Ex. 9 bisulfite- bisulfite- with xs Ex. 9bisulfite- bisulfite- treated treated enzyme bisulfite- treated DNAtreated DNA DNA DNA treated (0.3 ng/μL) (0.2 ng/μL) with xs with xs DNAenzyme enzyme (0.6 ng/ μL)

Each PCR was prepared as described in the MSP analysis describedelsewhere herein. MSP primer sets used were Mlh M, Mlh U, Dapk M, DapkU, Mgmt M, Mgmt U, p16 M, and p16 U. When excess TaqGold polymerase wasused, an additional 24, (2 units) was added to the reaction.

The results show that extra enzyme in the Master Mix forced “mispriming”to occur (determined by subsequent sequencing). The 1/10, 1/20, and 1/30dilutions of the Microcon 100 purified bisulfite-treated gDNA stillprovided enough gDNA template for MSP for almost all of the reactions.Successful PCR was seen even when only 0.2 ng of gDNA was used in MSP.Although there were two data points that “dropped out,” overall the dataare excellent. Thus, it appears that as little as 0.2 ng of DNA can beused with successful PCR yields.

Studies Using Additional Templates

The studies above included only unmethylated DNA. The followingexperiments include side by side comparisons of methylated tounmethylated gDNA. Five additional “control” reactions were evaluatedwith both methylated and unmethylated gDNA. These samples contained 1 μLDNA from Coriell or 3 μL DNA from the Intergen p16 kit (each about 300ng DNA), 35 μL water, 104, 20% Et₄NOH, and 854, 2M magnesium bisulfiteand heated at 50° C. for about 4 hours. The samples were Coriell #34 and#35, and DNA from Intergen's p16 “kit,” p16U, p16M, and a universallymethylated gDNA. Each was incubated at 50° for four hours and subjectedto the size-exclusion clean-up method, using a Microcon 100 filtrationunit received 50 μL TE. One μL was used in MSP with the following MSPprimer pairs: MLH M, MLH U, Dapk M, Dapk U, Mgmt M, Mgmt U, p16M, andp16 U. Excellent MSP yields were obtained as evidence by an amplicon ofthe correct size, several of the PCRs were subjected to directsequencing.

The same bisulfite treated samples were additionally analyzed bysequencing with other primers for different gene targets: E2F2, Frap,Xpd, CDKN1C, Ral GDS, Etsl, Cdhl, Apcl, Esrl, MLh1, and CMyc. Theseprimers are:

E2F2FwdFam FAM-GGTTTGGGGAATATATTGTTGGG E2F2RevCTTAAAAAAACAACCACACCTACTATTAATACC Cdh1FwdFamFAM-TGTGTTTGTAGGAGTTTGTGTTTGTG Cdh1Rev CTCCAAAATCCTCCAAACCC Frap1FwdFamFAM-GATTGGTTTTTAGGGTTGGGAA Frap1Rev TCCCCTAACCCCCCCTC XpdFwdFamFAM-GGGTTTGATTAATATTTAATTTTGGTAGG XpdRev TCAATCCACTAAAACACAACCAATCCDKN1CfwdFam FAM-GTTTTATAGGTTAAGTGTGTTGTGTT CDKN1CrevCACTAATACTAAAAAAATCCCACAAAC RalGDSFwdFamFAM-GGGTTTTATAGTTTTTGTATTTAGGTTTTTATTG RalGDSRevCAACTCAATAAACTCAAACTCCCC ID2FwdFam FAM-GAAGGTGAGTAAGATGGAAATTTTGTAGTAID2Rev ACTAACAATTTCACACACAACTCAATCTAC ApclFwdFamFAM-AGGGAAAATTGGAGTAGGAGGTT ApclRev ACTCAACTCCCCAAAACTATCCTTAAEsr1FwdFam FAM-TGGGAGATTAGTATTTAAAGTTGGAGG Esr1RevCCTTAAATCTAATACAATAAAACCATCCC Ets1FwdFam FAM-GGGAATTTGAGATTTTTGGGAAGEts1Rev CCCAACTACCAACAACATCCC Mlh1FwdFamFAM-GTAGTTTTTTTTTTAGGAGTGAAGGAGGT Mlh1Rev CCCTACTCTTATAACCTCCCACAAATCMycFwdFam FAM-GGGAGGTTATTTTGTTTATTTGGG CMycRevCCAAAACCCAAAAAACAATTAACAC

Comparison of Magnesium Bisulfite/Et₄NOH to Sodium Bisulfite ProtocolAfter 6 Hour and 15 Hour Reaction Times

A direct comparison between samples prepared according to the sodiumbisulfite protocol (J. Herman, Proc. Natl. Acad. Sci. 93, 9821-26(1996)) and the magnesium bisulfite protocol as described in Ex. 10 wasconducted, using Et₄NOH, NaOH, and no additive. Two plates were set up,one for a 6 hour analysis and the other for a 15 hour analysis. Bothreactions took place at 50° C. Only an unmethylated gDNA sample wasinvestigated. All reaction products were purified by the size-exclusionclean-up procedure described above and recovered in a final volume of504, of TE MSP was used to analyze the converted DNA. The sodiumbisulfite procedure provided gDNA that gave excellent results in MSP.The magnesium bisulfite converted gDNA gave much weaker signals in MSPthan the sodium bisulfite converted DNA. However, the magnesiumbisulfite/Et₄NOH differs from the sodium bisulfite protocol insignificant ways. The magnesium bisulfite/Et₄NOH was achieved without apre-denaturation step, much lower concentration of bisulfite, noexacting pH control, no antioxidant, and reagents were “off the shelf”and not freshly prepared.

The size-exclusion purification worked well on the sodium bisulfitesamples as well as the magnesium bisulfite samples.

Direct Comparison of Magnesium Bisulfite with Et₄NOH and SodiumBisulfite Reactions on Both Methylated and Unmethylated gDNA

The 1.3M (final concentration) magnesium bisulfite with Et₄NOH reactionwas compared to the samples treated with sodium bisulfate, according tothe known method. The magnesium bisulfite recipe was 1 μL Coriell or 3μL Intergen, 32 or 34 μL water, 10 μL 20% Et₄NOH, 85 μL 2M magnesiumbisulfite. Two methylated samples and two unmethylated samples werecompared in side by side reactions with sodium bisulfite and themagnesium bisulfite. These were allowed to react for 6 and 15 hours at50° for a total of sixteen (16) samples processed. Purification by thesize-exclusion process described above was performed. In this verythorough comparison, the 16 samples, purified by Microcon 100, were allanalyzed by sequencing. (The sequencing analysis allows for all cytosinein a given region to be analyzed for completeness of the bisulfiteconversion to uracil.)

Eleven different primer sets for specific gene targets were used: E2F2,FRAP, XPD, CDKN, Ral DGS, IDT, CDH1, APC1, and ESR. The results showedthat the methods disclosed herein are viable alternatives to the sodiumbisulfite reaction. The studies herein utilized 2M magnesium bisulfitesolution, which is diluted in the sample to about 1.3M. Use of a moreconcentrated magnesium bisulfite solution would yield higher bisulfiteconcentration for conversion, while still keeping reaction volumes to aminimum. Such increased bisulfite concentration in the reaction mixturecould easily be employed, and would be expected to enhance PCR yields.The optimization of such reaction parameters, including volume and/orconcentration of magnesium bisulfite solution, temperature, pH and otherreaction conditions are expected to lead to more complete conversion,and are well within the skill of the art.

Evaluation of HPLC Model System

A model system using a synthetic, four base oligonucleotide, ATCG, wasemployed to determine the rate of cytosine to uracil conversion by HPLC.Samples contained Et₄NOH, no additive, tetramethyl ammonium chloride(TMAC), or guanidine HCl in the magnesium bisulfite reaction. Thecomposition of the samples of Examples 19-22 is shown in Table 5 below.

TABLE 5 Ex. 19 Ex. 20 Ex. 21 Ex. 22 ATCG 2.5 μL ATCG 2.5 μL ATCG 2.5 μLATCG 2.5 μL 2M Mg(HSO₃) 2M Mg(HSO₃) 2M Mg(HSO₃) 2M Mg(HSO₃) 16.3 μL 16.3μL 16.3 μL 16.3 μL (~1.3M final) (~1.3M final) (~1.3M final) (~1.3Mfinal) 20% Et₄NOH 2 — TMAC 1.25 μL 3M Guanidine HCl μL 0.83 μL (0.1Mfinal) Water 4.2 μL Water 6 μL Water 4.95 μL Water 5.4uL pH ~ 4 pH ~3 pH~3 pH ~3-4

Each sample was heated at 50° for about 28 minutes. The pH of thesamples was measured after heating with pH paper, and is thereforeapproximate. All samples reacted comparably to each other, with theEt₄NOH reaction performing slightly better than the others.

Another HPLC study was conducted evaluating Et₄NOH, NaOH, Et₄NCl, andguanidine thiocyanide as catalysts. The composition of the samples(Examples 23-26) is shown in Table 6, below.

TABLE 6 Ex. 23 Ex. 24 Ex. 25 Ex. 26 ATCG 2.5 ATCG 2.5 μL ATCG 2.5 μLATCG 2.5 μL μL 2M Mg(HSO₃)₂ 2M Mg(HSO₃)₂ 2M Mg(HSO₃)₂ — 16.3 Ml 16.3 μL16.3 μL (~1.3M final) (~1.3M final) (~1.3M final) 20% Et₄NOH 2 5M NaOH0.5 2M Et₄NCl 2 μL 2M guanidine μL μL thiocyanide 16.3 μL Water 4.2 μLWater 5.7uL Water 4.2 μL Water 6.2 uL pH ~3 pH ~3 pH ~2 pH ~4

Each sample was heated at 50° for about 32 minutes. pH was measuredafter heating with pH paper, and is therefore approximate. Each of thereactions compared favorably to each other, with the exception of theguanidine thiocyanide reaction, which did not react at all. The Et₄NOHreaction provided the beast results, with the NaOH reaction being nearlyas effective. The Et₄NCl reaction was useable, but was less effectivethan either the NaOH reaction or the Et₄NOH reaction. Thus, pH may beimportant in the reaction.

Applicants have also discovered that, although they see significantbenefits in using magnesium bisulfite instead of sodium bisulfite,significant improvements may also be seen, regardless of which bisulfiteis used, with the modified purification processes discussed herein.Processes for purification are further discussed in the applicationentitled “Improved Bisulfite Method” (U.S. application Ser. No.60/498,996 filed Aug. 29, 2003, and also application Ser. No. 60/520,941(5109P2) having the same title and filed Nov. 17, 2003), assigned to theAssignee hereof, and which is incorporated by reference in its entirety.One embodiment of that process uses a Microcon 100 (Millipore), orsimilar, size-exclusion device. According to one embodiment of thatmethod, the sample and 200 μL of water was added to the Microcon 100device, and the sample was then spun in the device at approximately 2800RPM for about 8 minutes (as per manufacturers recommendation). Theresultant filtrate was removed. Two subsequent washes with about 300 μLwater, each spun at about 2800 RPM for 8 minutes followed. After each,the filtrate was again removed. About 300 μL 0.1N NaOH was added andspun at approximately 2800 RPM for about 8 minutes. Again, the filtratewas removed. After addition of about 300 μL of water, the sample wasspun in the device at 2800 RPM for about 6-8 minutes. The filtrate wasremoved and about 50 μL TE buffer was added. After about 5 minutesbefore it was inverted to collect the purified DNA sample in acentrifuge. Approximately 60 μL were collected.

While the above-described methods of PCR and sequencing are currentlypreferred, they are not the only methods useable. The present inventionis not limited to these any particular embodiments, or any of theexamples above. Rather, other variants of these methods will be apparentto those skilled in the art and are within the scope and spirit of theinvention disclosed herein.

1. A method for converting cytosine to uracil comprising the steps of:providing a nucleic acid comprising at least one cytosine nucleobase;and reacting the nucleic acid with a bisulfite ion, in the presence of aquaternary amine catalyst having the Formula:

or a derivative thereof, wherein: R₁, R₂, R₃ and R₄ are eachindependently C₁-C₄ alkyl; and Z is selected from halides and OH.
 2. Themethod of claim 1, wherein the nucleic acid is gDNA and further whereina step of predenaturation of the gDNA prior to the step of reacting thegDNA with the bisulfite ion is not performed.
 3. The method of claim 1,wherein the quaternary amine catalyst comprises at least one ofquaternary methyl ammonium bromide, tetraethylammonium hydroxide,tetraethylammonium chloride, tetrabutylammonium chloride andtetrabutylammonium bromide.
 4. The method of claim 1, wherein thecatalyst comprises tetraethylammonium hydroxide.
 5. The method of claim4 wherein the tetraethylammonium hydroxide is provided as anapproximately 20% solution.
 6. The method of claim 1 wherein thebisulfite ion comprises magnesium bisulfite.
 7. The method of claim 6wherein the magnesium bisulfite is provided as an approximately 2Msolution.
 8. The method of claim 7 wherein the final concentration ofthe magnesium bisulfite in the reaction mixture is approximately 1.3M.9. The method of claim 1 wherein the reaction is performed at about 50°C. for about 4 to about 15 hours.
 10. The method of claim 1, furthercomprising treating the product of the reaction of the nucleic acid andthe bisulfite ion with NaOH.
 11. The method of claim 11, wherein theNaOH is provided as an approximately 0.1M solution.
 12. A kit for use inconversion of cytosine to uracil in a nucleic acid comprising: magnesiumbisulfite; and a quaternary amine catalyst.
 13. The kit of claim 12,wherein the magnesium bisulfite is provided as an approximately 2Msolution.
 14. The kit of claim 12 wherein the quaternary amine catalystcomprises at least one of quaternary methyl ammonium bromide, tetraethylammonium hydroxide, tetraethyl ammonium chloride, tetrabutyl ammoniumchloride and tetrabutyl ammonium bromide.
 15. The kit of claim 14wherein the quaternary amine catalyst comprises tetraethylene ammoniumhydroxide.
 16. The kit of claim 15 wherein the tetraethyl ammoniumhydroxide is provided as an approximately 20% solution.
 17. The kit ofclaim 12 further comprising NaOH, wherein the NaOH is provided as anapproximately 0.1M solution.
 18. The kit of claim 12 further comprisinga nucleotide polymerase and one or more primers for sequencing oramplification.
 19. The kit of claim 12 further comprising one or morereaction vessels.
 20. The kit of claim 12 further comprising prepackagedmaterials sufficient to convert cytosine to uracil in one or morenucleic acid samples containing at least one cytosine nucleobase.